Multiple Assay Device

ABSTRACT

Disclosed is an assay device for the determination of analyte in a liquid sample over an extended concentration range comprising a first assay and a second assay, wherein the first assay for an analyte comprises a first flow-path having a sole detection zone capable of immobilising a labelled binding reagent and the second assay for said analyte comprises a second flow-path having a sole detection zone capable of immobilising a labelled binding reagent, wherein the presence of labelled binding reagent at the detection zones provides an indication of the presence and/or extent of analyte in said liquid sample.

RELATED APPLICATIONS

The present application is a continuation of U.S. Utility patent application Ser. No. 12/202,639 filed Sep. 2, 2008, which claims benefit of priority to U.S. Provisional Patent Application No. 60/991,531, filed on Nov. 30, 2007; Great Britain Application 0706906.5, filed Apr. 10, 2007; and Great Britain Application 0717043.4, filed Sep. 1, 2007, the contents of which are incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

The present invention relates to an assay device, kit and method for determining the presence or concentration of an analyte over an extended concentration range.

BACKGROUND OF THE INVENTION

Simple lateral flow immunoassay devices have been developed and commercialised for detection of analytes in fluid samples, see for example EP291194. Such devices typically comprise a porous carrier comprising a dried mobilisable labelled binding reagent capable of binding to the analyte in question, and an immobilised binding reagent also capable of binding to the analyte provided at a detection zone downstream from the labelled binding reagent. Detection of the immobilised labelled binding at the detection zone provides an indication of the presence of analyte in the sample.

Alternatively, when the analyte of interest is a hapten, the immunoassay device may employ a competition reaction wherein a labelled analyte or analyte analogue competes with analyte present in the sample for an immobilised binding reagent at a detection zone. Alternatively the assay device may employ an inhibition reaction whereby an immobilised analyte or analyte analogue is provided a detection zone, the assay device comprising a mobilisable labelled binding reagent for the analyte.

A sandwich immunoassay is often the assay of choice when detecting analytes. However, a sandwich assay is not always possible, for example in the case of small molecules such as haptens which may not be large enough to allow the simultaneous binding thereto of two different binding partners. A dose-response curve prepared using a typical lateral flow device employing a sandwich immunoassay shows increasing levels of signal with increasing analyte up to the point where at higher analyte levels the curve tends to plateau. At yet higher analyte levels, the signal begins to decrease due to preferential capture at the detection zone of analyte which has not yet bound to labelled reagent. This phenomenon is known as the hook effect. Thus sandwich immunoassays exhibit a limited assay range due to the fact that the signal amount or intensity observed at higher analyte levels may be the same, or even less, than that observed at lower analyte levels.

A competition or inhibition assay typically provides a high signal at zero or low levels of analyte. At increasing levels of analyte the signal level may still be high depending upon the amount of labelled binding species present compared to the amount of analyte. At still increasing levels of analyte, the signal starts to decrease as unbound analyte either competes with labelled analyte or analyte analogue for the immobilised binding reagent or binds to labelled binding reagent, lowering binding of the unlabelled binding reagent at the detection zone.

Thus the above assay methods are not suitable for measuring levels of analyte over an extended analyte range.

US2005/0112780 discloses an assay device and method for extending the dynamic detection range of assay devices comprising a flow through porous carrier comprising a detection zone and a compensation zone provided downstream from the detection zone. The detection involves a first binding reagent which binds a detection probe to generate a detection signal having an intensity proportional to the amount of analyte, and the compensation zone comprises a second capture reagent which binds a detection probe to generate a signal which is inversely proportional to the intensity of the detection signal. The assay may further comprise a third calibration zone which generates a signal. The first binding reagent may be selected from a group including an antigen, hapten or streptavidin. The first and second binding reagents may be chosen from a number of species including an antigen, hapten or streptavidin.

US2004/0197820 discloses a flow through porous carrier assay device for reducing the hook effect comprising a detection zone wherein the device may include a downstream calibration zone.

US2006/0019404 discloses an assay device with an extended dynamic range comprising a lateral flow test-strip comprising a plurality of detection zones with a progressively decreased sensitivity to analyte concentration. The assay device may comprise two carriers each having a plurality of detection zones. The amount of label/signal present at the plurality of detection zones is detected to determine the analyte concentration.

EP462376 discloses an assay device comprising a capture site and a conjugate recovery site wherein the conjugate recovery site receives and binds said conjugate or conjugate complexes which migrate through said capture site and wherein immobilised conjugate at both the conjugate recovery site and capture site is detected to determine the amount of the analyte of interest.

The present inventors have shown that for assay devices wherein multiple detection zones for the detection of an analyte are provided on the same porous carrier, binding at an upstream detection zone may change the binding characteristics at a downstream detection zone and that any variation in binding at an upstream detection zone may cause a compounded variation of binding at a downstream detection zone. This is especially so at higher analyte concentration levels and can give rise to poor assay precision. Furthermore, it has been found that cross-binding may occur between the respective binding reagents present in the detection zones during running of the test and cross-binding has also been observed during manufacture of the devices and whilst they are stored in the dry state. This was shown to have an impact on the levels of assay precision and sensitivity. These problems do not appear to have been recognised previously in the prior art.

It is an object to provide an improved assay device, kit and method for extending the analyte range of an assay.

SUMMARY OF THE INVENTION

An assay device for determining the presence or concentration of an analyte such as hCG in a liquid sample over an extended concentration range is provided. In one embodiment, the device comprises a first flow-path having a sole detection zone capable of immobilising a labelled binding reagent and a second flow-path having a sole detection zone capable of immobilising a labelled binding reagent, wherein the presence of labelled binding reagent at the detection zones provides an indication of the presence or concentration of analyte in said liquid sample.

In another embodiment, an assay device may comprise a first flow-path and a second flow-path wherein said first flow-path comprises a porous carrier having a detection zone comprising an immobilised binding reagent for an analyte and provided upstream from the detection zone is a mobilisable labelled binding reagent for said analyte; and wherein said second flow-path comprises a detection zone comprising an immobilised binding reagent for the analyte and a mobilisable labelled binding reagent upstream from the detection zone for the analyte and a scavenger reagent for said analyte, wherein detection of the presence of labelled binding reagent at the detection zone provides an indication of the presence or concentration of analyte in said liquid sample.

In another aspect, the first flow-path is capable of providing an indication of the level of analyte in a first concentration range and the second flow-path is capable of providing an indication of the level of analyte in a second concentration range. The assay device according is capable of providing an indication of the level of analyte with respect to one or more thresholds. The first flow-path of the device may define a sandwich assay, and the second flow-path may define a competition or inhibition assay.

The first flow-path may comprise a mobilisable labelled binding reagent for the analyte provided upstream from a detection zone, wherein the detection zone comprises an immobilised binding reagent for the analyte and wherein the second flow-path comprises a mobilisable binding reagent for the analyte provided upstream from provided upstream from a detection zone, said detection zone comprising an immobilised analyte or analyte analogue for the mobilisable binding reagent. The first flow-path may define a high analyte sensitivity assay, and the second flow-path defines a low analyte sensitivity assay.

Assay kits and methods for determining the presence or concentration of an analyte in a liquid sample comprising an assay device comprising the devices are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

Aspects of the invention are further illustrated by reference to the following figures:

FIG. 1 illustrates typical signal responses that are observed for a typical assay compared with that of a typical competition assay.

FIG. 2 illustrates plots of signal intensity vs hCG concentration for Example 1 and Comparative Example 1.

FIG. 3 illustrates a plot of signal intensity vs hCG concentration for the assay device according to Example 2.

FIG. 4 illustrates the effect of varying the amounts of scavenger antibody for the assay device according to Example 2.

FIG. 5 illustrates the effect of varying latex spray positions to reduce a hook effect using a device as described herein.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise above, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Where a term is provided in the singular, the inventor also contemplates the plural of that term. The nomenclature used herein and the procedures described below are those well known and commonly employed in the art.

The singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

As used herein, the term “analyte” generally refers to a substance to be detected. The term “analyte” includes, but is not limited to, toxins, organic compounds, proteins, peptides, microorganisms, bacteria, viruses, amino acids, nucleic acids, carbohydrates, hormones, steroids, vitamins, drugs (including those administered for therapeutic purposes as well as those administered for illicit purposes), pollutants, pesticides, and metabolites of or antibodies to any of the above substances. The term “analyte” also includes any antigenic substances, haptens, antibodies, macromolecules, and combinations thereof. Specific examples of analytes include but are not limited to ferritin; creatinine kinase MB (CK-MB); digoxin; phenytoin; phenobarbitol; carbamazepine; vancomycin; gentamycin; theophylline; valproic acid; quinidine; luteinizing hormone (LH); follicle stimulating hormone (FSH); estradiol, progesterone; C-reactive protein; lipocalins; IgE antibodies; cytokines; vitamin B2 micro-globulin; glycated hemoglobin; cortisol; digitoxin; N-acetylprocainamide (NAPA); procainamide; antibodies to rubella, such as rubella-IgG and rubella IgM; antibodies to toxoplasmosis, such as toxoplasmosis IgG (Toxo-IgG) and toxoplasmosis IgM (Toxo-IgM); testosterone; salicylates; acetaminophen; hepatitis B virus surface antigen (HBsAg); antibodies to hepatitis B core antigen, such as anti-hepatitis B core antigen IgG and IgM (Anti-HBC); human immune deficiency virus 1 and 2 (HIV 1 and 2); human T-cell leukemia virus 1 and 2 (HTLV); hepatitis Be antigen (HBeAg); antibodies to hepatitis Be antigen (Anti-HBe); influenza virus; thyroid stimulating hormone (TSH); thyroxine (T4); total triiodothyronine (Total T3); free triiodothyronine (Free T3); carcinoembryoic antigen (CEA); lipoproteins, cholesterol, and triglycerides; and alpha fetoprotein (AFP). Drugs of abuse and controlled substances include, but are not intended to be limited to, amphetamine; methamphetamine; barbiturates, such as amobarbital, secobarbital, pentobarbital, phenobarbital, and barbital; benzodiazepines, such as librium and valium; cannabinoids, such as hashish and marijuana; cocaine; fentanyl; LSD; methaqualone; opiates, such as heroin, morphine, codeine, hydromorphone, hydrocodone, methadone, oxycodone, oxymorphone and opium; phencyclidine; and propoxyhene.

“Antigen” shall mean any compound capable of binding to an antibody, or against which antibodies can be raised.

“Antibody” shall mean an immunoglobulin having an area on its surface or in a cavity that specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of another molecule. The antibody can be polyclonal or monoclonal. Antibodies may include a complete immunoglobulin or fragments thereof, which immunoglobulins include the various classes and isotypes, such as IgA (IgA1 and IgA2), IgD, IgE, IgM, and IgG (IgG1, IgG2, IgG3, and IgG4) etc. Fragments thereof may include Fab, Fv and F(ab′)₂, Fab′, and the like. Antibodies may also include chimeric antibodies made by recombinant methods.

A “binding reagent” refers to a member of a binding pair, i.e., two different molecules wherein one of the molecules specifically binds with the second molecule through chemical or physical means. The two molecules are related in the sense that their binding with each other is such that they are capable of distinguishing their binding partner from other assay constituents having similar characteristics. The members of the specific binding pair (“sbp”) are referred to as ligand and receptor (antiligand), sbp member and sbp partner, and the like. In addition to antigen and antibody specific binding pair members, other specific binding pairs include biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, a peptide sequence and an antibody specific for the sequence or the entire protein, polymeric acids and bases, dyes and protein binders, peptides and specific protein binders (e.g., ribonuclease, S-peptide and ribonuclease S-protein), and the like. Furthermore, specific binding pairs can include members that are analogues of the original specific binding member, for example an analyte-analogue or a specific binding member made by recombinant techniques or molecular engineering. A molecule may also be a binding pair member for an aggregation of molecules; for example an antibody raised against an immune complex of a second antibody and its corresponding antigen may be considered to be a binding pair member for the immune complex. In addition to antigen and antibody binding pair members, other binding pairs include, as examples without limitation, biotin and avidin, carbohydrates and lectins, complementary nucleotide sequences, complementary peptide sequences, effector and receptor molecules, enzyme cofactors and enzymes, enzyme inhibitors and enzymes, a peptide sequence and an antibody specific for the sequence or the entire protein, polymeric acids and bases, dyes and protein binders, peptides and specific protein binders (e.g., ribonuclease, S-peptide and ribonuclease S-protein), and the like. Furthermore, specific binding pairs can include members that are analogues of the original specific binding member.

The terms “comprise” and “comprising” is used in the inclusive, open sense, meaning that additional elements may be included.

The term “flow-path” for the purposes of this invention refers to a substrate that is able to convey a liquid from a first position to a second position and may be for example a capillary channel, a microfluidic pathway, or a porous carrier such as a lateral flow porous carrier. The porous carrier may comprise one or a plurality of porous carrier materials which may overlap in a linear or stacked arrangement or which are fluidically connected. The porous carrier materials may be the same or different. A first flow path of a device provides for a first assay, and a second flow path of the device provides for a second assay.

“Label” when used in the context of a labelled binding reagent, refers to any substance which is capable of producing a signal that is detectable by visual or instrumental means. Various labels suitable for use in the present invention include labels which produce signals through either chemical or physical means, such as being optically detectable. Such labels include enzymes and substrates, chromogens, catalysts, fluorescent compounds, chemiluminescent compounds, electroactive species, dye molecules, radioactive labels and particle labels. The analyte itself may be inherently capable of producing a detectable signal. The label may be covalently attached to the binding reagent.

“Labelled binding reagent” refers to any substance comprising a detectable label attached to a binding reagent. The attachment may be covalent or non-covalent. The label provides a detectable signal that is related to the presence or amount of analyte in the fluid sample. Various labels suitable for use include labels which produce signals through either chemical, biochemical or physical means. Such labels can include enzymes and substrates, chromogens, catalysts, fluorescent compounds, chemiluminescent compounds, and radioactive labels. Other suitable labels include colloidal metallic particles such as gold or silver, colloidal non-metallic particles such as selenium or tellurium, dyed or colored particles such as a coloured polymer such as polystyrene, a stained microorganism or dyesols, organic polymer latex particles and liposomes, colored beads or electrochemically detectable species. Of the above, colored polymer particles and colloidal gold are preferred.

The term “non-labelled binding reagent” for the purposes of this invention refers to a binding reagent that is either not labelled with a detectable label or is labelled with a label which is not detected in order to determine the analyte.

“Porous carrier” refers to a porous body capable of transporting fluid sample.

The term “sample” refers to any sample potentially containing an analyte. For example, a sample may be a bodily fluid such as blood, urine, mucous or saliva, or a respiratory sample, such as a nasopharyngeal wash or aspirate, nasal swab, nasopharyngeal swab, nasal wash, throat swab, transtracheal aspirate, bronchoalveolar lavage, elution buffer used to wash a respiratory sample, etc.

For the purpose of this application the term “scavenger binding reagent” denotes an additional binding reagent capable of binding analyte and the term “scavenger” is used merely to distinguish the binding reagents from the other binding reagents present in the device.

According to a first aspect, the invention provides an assay device for the determination of analyte in a liquid sample over an extended concentration range comprising a first assay and a second assay, wherein the first assay for an analyte comprises a first flow-path having a sole detection zone capable of immobilising a labelled binding reagent and the second assay for said analyte comprises a second flow-path having a sole detection zone capable of immobilising a labelled binding reagent, wherein the presence of the labelled binding reagent at the detection zones provides an indication of the presence and/or concentration of analyte in said liquid sample.

The first assay may provide an indication of the level of analyte in a first concentration range and the second assay may provide an indication of the level of analyte in a second concentration range.

The first and second concentration ranges differ from each other. The first and second concentration ranges may overlap so as to provide a continuous concentration range.

The assay device may be capable of providing an indication of the level of analyte with respect to one or more thresholds. The assay device may provide an indication of the level of analyte below or above a plurality of thresholds. For example the number of thresholds may be two three, four, five or greater.

The first and second assays may either independently or together provide an indication of the level of analyte within a certain range.

According to an embodiment, the first assay provides an indication of the level of analyte of less than or equal to a first threshold and the second assay provides an indication of the level of analyte of above or equal to a second threshold. The first and second assays together provide an indication of the level of analyte of greater than the first threshold but less than the second threshold.

A non-labelled binding reagent for the analyte or an analyte analogue may be provided in an immobilised form at a detection zone. The non-labelled binding reagent may be chosen from a binding reagent for the analyte of interest, an analyte or analyte analogue, depending upon whether the assay is a sandwich type assay or a competition type assay. Similarly the labelled binding reagent may comprise a labelled binding reagent for the analyte of interest, a labelled analyte or labelled analyte analogue.

Alternatively a reagent may be provided in an immobilised form at the detection zone that is capable of binding a labelled binding reagent-analyte- non-labelled binding reagent complex. For example the binding reagent may be conjugated or otherwise joined to binding species such as biotin, the reagent immobilised at the detection zone being a complementary binding partner such as streptavidin.

The non-labelled binding reagent may be provided in a mobilisable form which is capable of immobilising a labelled binding reagent-analyte complex at a detection zone. For example the non-labelled binding reagent may be attached to particle such as agarose and the detection zone may comprise a filter of dimensions smaller than the particle, but larger than the size of the labelled binding reagent, such that the filter is able to trap the any labelled binding reagent-analyte-non-labelled binding reagent complex present, any labelled binding reagent that is not complexed to the capture reagent being able to pass through the filter.

The first and/or second assay may comprise a labelled binding reagent provided in a mobilisable form upstream from the detection zone reagent in the dry state prior to use of the device.

The first and second assays may each comprise a mobilisable labelled binding reagent provided upstream from an immobilised non-labelled binding provided at each detection zone.

The assay device may comprise more than two assays, each capable of detecting the analyte at a particular concentration range or above or below one or more thresholds.

The first and second assays may individually or together provide an indication of the particular level of analyte, or whether the analyte is above or below a certain threshold.

The assay device may have a common sample application region that fluidically connects the plurality of flow-paths. Thus a fluid sample applied to sample application region of the device is able to travel along the flow-paths of the respective assays to the respective detection zones. The sample application region may comprise a porous sample receiver. In the case where the mobilisable labelled binding reagent is the same for both assays it may be provided at the sample application region.

As an alternative to providing the first and second assays within a single assay device, the assays may be provided as separate assay devices, the results from the respective devices when taken together being capable of providing an indication or measurement of the level of analyte.

Thus according to a second aspect, the invention provides an assay kit for the determination of an analyte over an extended concentration range comprising a first assay device and a second assay device, wherein the first and second assay devices comprise respectively first and second assays according to the first aspect of the invention.

According to a third aspect, the invention provides a method for the determination of an analyte over an extended analyte range comprising the steps of:

-   -   a) adding a liquid sample to a first assay comprising a         mobilisable labelled binding reagent provided upstream from a         sole detection zone and to a second assay comprising a         mobilisable labelled binding reagent provided upstream from a         sole detection zone, said detection zones being capable of         immobilising labelled binding reagent, and wherein detection of         labelled reagent at the detection zones provides an indication         of the concentration and/or presence of an analyte in the liquid         sample.     -   b) reading the result of the assay.

In the case where the level of analyte is known to vary as a function of time, for example the pregnancy hormone hCG, the assay device may provide a time-based indication to the user, such as the duration of pregnancy in units of days or weeks.

The first and second flow paths may be provided on separate substrates or they may be provided on a common substrate such that liquid being conveyed along a flow-path of the first assay is not able to cross over to the flow-path of the second assay. For example, the first and second assays may be provided on the same porous carrier such that the first and second flow-paths are isolated from each other. This may be achieved for example by laser cutting parts of the porous carrier to make it non-porous, thus separating the first and second flow-paths. As yet a further alternative, the first and second detection zones may be provided on the same flow-path in substantially a side by side arrangement, such that neither is provided downstream from the other.

In particular the flow-path may be a lateral flow porous carrier. Suitable materials that may be employed as a porous carrier include nitrocellulose, acetate fibre, cellulose or cellulose derivatives, polyester, polyolefin or glass fibre. The porous carrier may comprise nitrocellulose. This has the advantage that a binding reagent can be immobilised firmly without prior chemical treatment. If the porous solid phase material comprises paper, for example, the immobilisation of the antibody in the second zone needs to be performed by chemical coupling using, for example, CNBr, carbonyldiimidazole, or tresyl chloride.

The label may comprise a particle such as gold, silver, colloidal non-metallic particles such as selenium or tellurium, dyed or coloured particles such as a polymer particle incorporating a dye, or a dye sol. The dye may be of any suitable colour, for example blue. The dye may be fluorescent. Dye sols may be prepared from commercially-available hydrophobic dyestuffs such as Foron Blue SRP (Sandoz) and Resolin Blue BBLS (Bayer). Suitable polymer labels may be chosen from a range of synthetic polymers, such as polystyrene, polyvinyltoluene, polystyrene-acrylic acid and polyacrolein. The monomers used are normally water-insuluble, and are emulsified in aqueous surfactant so that monomer mycelles are formed, which are then induced to polymerise by the addition of initiator to the emulsion. Substantially spherical polymer particles are produced. An ideal size range for such polymer particles is from about 0.05 to about 0.5 μm. According to an exemplary embodiment the label is a blue polymeric particle.

The liquid sample can be derived from any source, such as an industrial, environmental, agricultural, or biological source. The sample may be derived from or consist of a physiological source including blood, serum, plasma, interstitial fluid, saliva, sputum, ocular lens liquid, sweat, urine, milk, ascots liquid, mucous, synovial liquid, peritoneal liquid, transdermalexudates, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinal liquid, semen, cervical mucus, vaginal or urethral secretions and amniotic liquid. In particular the source is human and in particular the sample is urine.

A particular analyte is human chorionic gonadotropin (hCG). The analyte may have a sole binding region or epitope or may have more than one binding region. For example the analyte hCG comprises an alpha sub-unit identical to that of luteinising hormone (LH), follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH) and a beta sub-unit unique to hCG. Antibodies to the alpha and beta sub-units may be used to bind to hCG in a sandwich immunoassay format.

The assay device of the invention may be used to measure the presence of hCG or concentration over an extended range. The range may vary from between, for example, about 10 mIU to about 250,000 mIU, from between about 30 mIU to about 200,000 mIU, from between about 50 mIU to about 175,000 mIU, from between about 100 mIU to about 100,000 mIU, from between about 250 mIU to about 75,000 mIU, from between about 300 mIU to about 50,000 mIU, or from between about 500 mIU to about 25,000 mIU.

According to an embodiment, the device is able to measure the amount of hCG in the fluid sample and to indicate to the user, based upon stored reference values the duration of pregnancy in time based units. The device may also indicate whether the subject is pregnant or not, determined by whether the level of hCG is above or below a base threshold. The reference and threshold values are typically stored within the device as part of an algorithm. The base threshold may typically range from 10-25 mIU/ml. The base threshold may be, for example, 10, 15, 20 or 25 mIU/ml.

According to an embodiment, the first assay may provide either an indication of whether the subject is pregnant or not, based upon whether the level of hCG detected is respectively above or below a base threshold, and/or if pregnant, the level of hCG in a first range of less than or equal to a first threshold, the second assay provides an indication of the level of hCG in a second range of above or equal to a second threshold and wherein the first and second assays together provide an indication of the level of hCG in a third range of greater than the first threshold but less than the second threshold.

The first and/or second assay may further comprise a control zone to indicate that the reagents have mobilised and have been transported along the flow path and that the assay test has been carried out satisfactorily. The control zone is typically positioned downstream from the detection zone and may for example comprise an immobilised binding reagent for a labelled binding reagent. The labelled binding reagent may be present in a mobilisable form upstream from the control zone and detection zone. The labelled binding reagent may the same or different to the labelled binding reagent for the analyte.

The assay device may comprise a porous sample receiver in fluid connection with and upstream from the first and second flow-paths. The porous sample receiver may be provided within the housing or may at least partially extend out of said housing and may serve for example to collect a urine stream. The porous sample receiver may act as a fluid reservoir. The porous sample receiving member can be made from any bibulous, porous or fibrous material capable of absorbing liquid rapidly. The porosity of the material can be unidirectional (ie with pores or fibres running wholly or predominantly parallel to an axis of the member) or multidirectional (omnidirectional, so that the member has an amorphous sponge-like structure). Porous plastics material, such as polypropylene, polyethylene (preferably of very high molecular weight), polyvinylidene flouride, ethylene vinylacetate, acrylonitrile and polytetrafluoro-ethylene can be used. Other suitable materials include glass-fibre.

If desired, an absorbant “sink” can be provided at the distal end of the carrier material. The absorbent sink may comprise of, for example, Whatman 3MM chromatography paper, and should provide sufficient absorptive capacity to allow any unbound labelled binding reagent to wash out of the detection zone. As an alternative to such a sink it can be sufficient to have a length of porous solid phase material which extends beyond the detection zone.

Following the application of a binding reagent to a detection zone, the remainder of the porous solid phase material may be treated to block any remaining binding sites. Blocking can be achieved by treatment for example with protein (e.g. bovine serum albumin or milk protein), or with polyvinylalcohol or ethanolamine, or combinations thereof. To assist the free mobility of the labelled binding reagent when the porous carrier is moistened with the sample, the porous carrier may further comprise a material such as sucrose or lactose. Such material may be depositing for example as an aqueous solution in the region to which the labelled binding reagent is to be applied. Alternatively such material may be deposited upstream from or at the labelled binding reagent.

The nitrocellulose porous carrier may have having a pore size of at least about 1 micron, for example greater than about 5 microns, and for example about 8-12 microns.

The nitrocellulose porous carrier may be backed e.g. with a plastics sheet, to increase its handling strength. This can be manufactured easily by forming a thin layer of nitrocellulose on a sheet of backing material such as Mylar™.

The labelled binding reagent may be provided on a separate macroporous carrier material to that of the detection zone. The macroporous carrier material should be low or non-protein-binding, or should be easily blockable by means of reagents such as BSA or PVA, to minimise non-specific binding and to facilitate free movement of the labelled reagent after the macroporous body has become moistened with the liquid sample. The macroporous carrier material can be pre-treated with a surface active agent or solvent, if necessary, to render it more hydrophilic and to promote rapid uptake of the liquid sample. Suitable materials for a macroporous carrier include plastics such as polyethylene or glass-fibre. In the case that the labelled binding reagent is labelled with a detectable particle, the macroporous body may have a pore size at least ten times greater than the maximum particle size of the particle label. Larger pore sizes give better release of the labelled reagent. As an alternative to a macroporous carrier, the labelled binding reagent may be provided on a non-porous substrate provided upstream from the detection zone, said non-porous substrate forming part of the flow-path.

The first and/or second assays may comprise a glass-fibre macroporous carrier provided upstream from and overlapping at its distal end a nitrocellulose porous carrier.

The assay device or kit may further comprise one or more optical detection means such as a photodetector and one or more light sources such as an LED positioned so as to optically illuminate the detection zones and determine the presence and/or amount of labelled species present. The assay device may further comprise one or more of a power source, a computation means, a signal transduction means, an algorithm, a display means, a memory means and data in/out port. The assay device may comprise a housing which serves to house the first and second assays as well as other components of the device. The device may comprise stored threshold values.

The first and second assays may be provided for example in a side by side arrangement or in a face to face arrangement wherein one assay is provided above the other. A sole optical means may be arranged to detect both detection zones.

In addition to measuring the detection zones of the respective assays as well as the control zones where present, the optical means may also measure a reference zone, namely a portion of the flow-path which is free from binding reagent in the dry state.

The purpose of the reference zone is to provide a signal value against which the signal value obtained at the detection zone may be referenced. This takes into account any change in optical signal due to for example, the wetting of the porous carrier any differences in the optical absorption of the liquid sample.

An assay device for measuring the levels of analyte in a liquid sample comprising an optical detection means arranged to measure the intensity of light reflected from a detection zone, control zone and reference zone of an assay device is described in EP1484601.

The illumination process may be carried out sequentially such that device is able to know which from which zone light is being reflected from onto the photodetector. The assay strips may be positioned in a side by side arrangement and the photodetector and light sources positioned above the plane of the strips such that the detection control and reference zones are positioned towards the light sources and optical detectors.

The first assay differs from the second assay such that the respective assays are capable of measuring analyte at different levels.

For example the first and second assays may employ differing assay architectures, such as the first assay employing a sandwich binding reaction and the second assay employing a competition or inhibition reaction. The first assay may comprise a mobilisable labelled binding reagent for the analyte provided upstream from a detection zone, said detection zone comprising a non-labelled immobilised binding reagent for the analyte and the second assay may comprise a mobilisable binding reagent for the analyte provided upstream from an immobilised non-labelled binding reagent for the mobilisable binding reagent. Alternatively the second assay may comprise a mobilisable labelled analyte or analyte analogue reagent provided upstream from an immobilised non-labelled binding reagent for the analyte. The sandwich assay is the high sensitivity assay, namely it is capable of measuring analyte at a lower concentration range and the inhibition or competition assay is a low sensitivity assay, namely it is capable of measuring analyte at a higher concentration range.

The assay device may for example comprise first and second assays wherein the non-labelled binding reagent of the first assay differ from the non-labelled binding reagent of the second assay, and/or the labelled binding reagent of the first assay differs from the labelled binding reagent of the second assay. For example this may be a difference in concentration, or a difference in affinity for an analyte, analyte analogue or binding reagent. A high affinity binding reagent will have a higher analyte sensitivity than a lower affinity binding reagent. Similarly a low concentration of binding reagent will have a lower analyte sensitivity than a high concentration of binding reagent. The first and second assays may be varied in this way such that they are capable of determining an analyte at different concentration ranges.

Thus the assay device may comprise a high analyte sensitivity first assay comprising a mobilisable labelled binding reagent of a certain concentration or affinity provided upstream from a detection zone and a low analyte sensitivity second assay comprising a mobilisable labelled binding reagent having a lower concentration or affinity provided upstream from a detection zone. Alternatively or additionally, the first assay comprising an immobilised binding reagent at a detection zone of a certain concentration or affinity and a second assay may comprise an immobilised binding reagent at a detection zone having a lower concentration or affinity.

Another way to lower the sensitivity of an assay is to reduce the binding reagent to particle loading. Thus the assay device may comprise a first high analyte sensitivity assay and a second low analyte sensitivity assay wherein the first assay comprises a mobilisable particle labelled binding reagent provided upstream from a detection zone having a ratio of binding reagent to particle label and wherein the second assay comprises a mobilisable particle labelled binding reagent provided upstream from a detection zone having a lower ratio of binding reagent to particle label than that of the first assay.

A further way to lower the sensitivity of an assay is to provide a labelled binding reagent having a label of a low optical density. This may be achieved for example by provision of a polymer particle label having a low concentration of dye. Thus the assay device may comprise a first high analyte sensitivity assay and a second low analyte sensitivity assay wherein the first assay comprises a mobilisable particle labelled binding reagent provided upstream from a detection zone, said label having an optical density and wherein the second assay comprises a mobilisable particle labelled binding reagent provided upstream from a detection zone wherein the label has a lower optical density than that of the first assay.

Yet a further way to measure high analyte levels is to employ a non-particulate labelled binding reagent. High levels of analyte when measured by way of a sandwich binding assay require high levels of binding reagent. In the case wherein the label is a particle label, provision of high levels of analyte within or on the porous carrier can give rise to steric hindrance resulting in poor assay sensitivity. Conversely, at lower analyte levels, the use of a non-particle labelled binding reagent can give rise to a low signal due to the low optical density. However, at high analyte levels, non-particle labels may be present at sufficiently high levels to readily detected. Therefore the assay may comprise a first high analyte sensitivity assay comprising an optically detectable particle labelled binding reagent provided upstream from a detection zone and a second low analyte sensitivity assay comprising an optically detectable non-particle labelled binding reagent provided upstream from a detection zone. An example of a optically detectable non-particle label may be a dye. The dye may be fluorescent.

A yet further way to lower the sensitivity of the assay is to employ a porous carrier such as nitrocellulose having a higher flow rate. Thus the assay device may comprise a first high analyte sensitivity assay having a porous carrier having a flow-rate and a second low analyte sensitivity assay having a porous carrier having a higher flow rate than that of the first assay.

A further way to lower analyte sensitivity is to provide for a fast release of the labelled binding reagent from the porous carrier during contact with the liquid sample. Ways to increase the release of labelled binding reagent are for example to increase the levels of sugars or methylcellulose in the device.

A further way to lower analyte sensitivity is to employ a labelled binding reagent having a lower optical sensitivity. One way of achieving this to use a colour which is less sensitive to an optical detector.

Use of a Scavenger Reagent

A further way to lower the analyte sensitivity is to provide a scavenger binding reagent to bind to analyte. The scavenger binding reagent may be provided upstream from a detection zone and may be immobilised, mobilisable or both. The scavenger binding reagent may be provided at either the same region of the porous carrier as the mobilisable binding reagent, upstream from it or downstream from it. The scavenger binding reagent may bind to the same binding region of the analyte as the mobilisable labelled binding reagent or to a different region of the analyte than the labelled binding reagent. Either or both of the assays may employ a scavenger binding reagent and the scavenger binding reagents may differ from one another in terms of their concentration, affinity or both.

According to an embodiment, the assay device comprises a first assay comprising a first porous carrier comprising a mobilisable labelled binding reagent provided upstream from a detection zone and a second assay comprising a mobilisable labelled binding reagent provided upstream from a detection zone and a scavenger binding reagent also provided upstream from the detection zone of the second assay. The first assay may be the high analyte sensitivity assay and the second assay may be the low analyte sensitivity assay.

The scavenger reagent may be provided in a mobilisable form.

The scavenger reagent may have a different affinity for the analyte than the mobilisable labelled binding reagent of the second assay. In an exemplary embodiment, the scavenger binding reagent has a higher affinity for the analyte than the mobilisable binding reagent of the second assay. The amount scavenger binding reagent may be varied to change the sensitivity of the second assay to analyte concentration. Increasing the amount of scavenger binding reagent present lowers the sensitivity of the assay due to the fact that the scavenger binding reagent is able to bind more analyte, effectively lowering the proportion of labelled binding reagent that is able to bind to the detection zone. The amount of labelled binding reagent in the first and second assays may be varied. Increasing the amount of labelled binding reagent has the tendency to reduce the hook effect and the amount of labelled binding reagent present, especially in the lower sensitivity assay, may be varied depending upon the analyte range.

The scavenger binding reagent may be capable of binding to the same or a different analyte binding region. In an exemplary embodiment, the scavenger binding reagent is capable of binding to a different binding region of the analyte. In particular where the analyte to be determined is hCG, the scavenger binding reagent is capable of binding to the beta-subunit, and the mobilisable labelled binding reagent is capable of binding to the alpha-subunit.

According to an exemplary embodiment, the assay device comprises a first assay comprising a glass-fibre porous carrier material comprising a mobilisable particle—labelled binding reagent for an analyte and a nitrocellulose porous carrier material provided downstream from the glass-fibre porous carrier material having a detection zone comprising a immobilised non-labelled binding reagent for the analyte and a second assay comprising a glass-fibre porous carrier material comprising a mobilisable particle—labelled binding reagent for a first binding region of the analyte and a mobilisable scavenger binding reagent for a second binding region of the analyte and a nitrocellulose porous carrier material provided downstream from the glass-fibre porous carrier material having a detection zone comprising an immobilised non-labelled binding reagent for the second binding region of the analyte.

It will be appreciated that the above ways to alter the assay sensitivity of an assay are not be exhaustive. The assay device may comprise one of more the above features to affect assay sensitivity. The particular assay architecture chosen would depend upon the analyte and its concentration range.

Other Embodiments

In addition to the embodiments, aspects and objects of the present invention disclosed herein, including the claims appended hereto, the following paragraphs set forth additional, non-limiting embodiments and other aspects of the present invention.

In one aspect the invention provides a assay device for the determination of analyte in a liquid sample over an extended concentration range comprising a first assay and a second assay, wherein the first assay for an analyte comprises a first flow-path having a sole detection zone capable of immobilising a labelled binding reagent and the second assay for said analyte comprises a second flow-path having a sole detection zone capable of immobilising a labelled binding reagent, wherein the presence of labelled binding reagent at the detection zones provides an indication of the presence and/or extent of analyte in said liquid sample. The first assay is capable of providing an indication of the level of analyte in a first concentration range and the second assay is capable of providing an indication of the level of analyte in a second concentration range. In certain embodiments the first and second concentration ranges overlap. The device is capable of providing an indication of the level of analyte with respect to one or more thresholds. In some embodiments, the first and/or second flow-path comprises a porous carrier, such as a lateral flow porous carrier.

In certain embodiments, the first and/or second assay comprises a mobilisable labelled binding reagent for the analyte provided upstream from a detection zone. In other embodiments, the detection zone of the first and/or second assay comprises an immobilised binding reagent for the analyte. Optionally, the first assay defines a sandwich assay and the second assay defines a competition or inhibition assay. In another embodiment, the first assay comprises a mobilisable labelled binding reagent for the analyte provided upstream from a detection zone, said detection zone comprising an immobilised binding reagent for the analyte and wherein the second assay comprises a mobilisable binding reagent for the analyte provided upstream from provided upstream from a detection zone, said detection zone comprising an immobilised analyte or analyte analogue for the mobilisable binding reagent. In other embodiments, the first assay comprises a mobilisable labelled binding reagent for the analyte provided upstream from a detection zone, said detection zone comprising an immobilised binding reagent for the analyte and wherein the second assay comprises a mobilisable labelled analyte or analyte analogue provided upstream from an immobilised binding reagent for the analyte.

In certain embodiments, the first assay is a high analyte sensitivity assay and the second assay is a low analyte sensitivity assay. In other embodiments, the first assay comprises a mobilisable labelled binding reagent for an analyte provided upstream from a detection zone, wherein the detection zone comprises an immobilised non-labelled binding reagent for the analyte; and wherein the second assay comprises a mobilisable labelled binding reagent for a first binding region of the analyte and a scavenger binding reagent for the analyte provided upstream from a detection zone, wherein said detection zone is capable of binding the labelled binding reagent. For example, the detection zone of the first and/or second assays comprises an immobilised binding reagent for the analyte. In another embodiment, for the second assay, the scavenger binding reagent has a higher affinity for the analyte than the mobilisable labelled binding reagent. Similarly, the mobilisable labelled binding reagent and the scavenger binding reagent of the second assay are for respectively a first and second binding region of the analyte, the detection zone comprising an immobilised binding reagent for a second binding region of the analyte. In a related embodiment, the mobilisable labelled binding reagent and the scavenger binding reagent of the second assay are provided in the same region. In another related embodiment, the scavenger binding reagent is provided in a mobilisable form. In certain embodiments, the mobilisable binding reagents and the scavenger reagent are provided on and/or in a first porous carrier material and the immobilised binding reagent provided at the detection zone is provided on and/or in a second porous carrier material. The first porous carrier material is, e.g., glass-fibre and the second porous carrier material is nitrocellulose.

In some embodiments, the analyte is hCG. For example, the first binding region of hCG is the alpha sub-unit and the second binding region is the beta sub-unit. Optionally, the binding reagents are antibodies, and/or the binding reagent is labelled with an optically detectable particle such as a coloured polymer label. In certain embodiments, the fluid sample is urine.

In some embodiments, the devices of the invention include a common sample application region which serves to supply liquid sample to both assays. In other embodiments, the device includes a housing wherein the first and second assays are provided within said housing.

In another aspect, the invention provides an assay device for determining the presence and/or extent of an analyte in a liquid sample comprising a first assay and a second assay wherein said first assay comprises a porous carrier having a detection zone comprising an immobilised binding reagent for an analyte and provided upstream from the detection zone is a mobilisable labelled binding reagent for said analyte; and wherein said second assay comprises a detection zone comprising an immobilised binding reagent for the analyte and provided upstream from the detection zone is a mobilisable labelled binding reagent for the analyte and a scavenger reagent for said analyte, wherein detection of the presence of labelled binding reagent at the detection zone provides an indication of the presence and/or extent of analyte in said liquid sample. In some embodiments, the first assay is a high sensitivity assay and the second assay is a low analyte sensitivity assay. In other embodiments, the analyte is hCG.

The invention provides devices in which the first assay either provides an indication of whether the subject is pregnant or not, based upon whether the level of hCG detected is respectively above or below a base threshold, and/or if pregnant, the level of hCG in a first range of less than or equal to a first threshold, the second assay provides an indication of the level of hCG in a second range of above or equal to a second threshold and the first and second assays together provide an indication of the level of hCG in a third range of greater than the first threshold but less than the second threshold. The invention also provides devices in which for the second assay, the scavenger binding reagent has a higher affinity for the analyte than the mobilisable labelled binding reagent.

In a further aspect, the invention provides an assay kit for determining the presence an/or extent of an analyte in a liquid sample comprising a first assay and a second assay wherein said first assay comprises a porous carrier having a detection zone comprising an immobilised binding reagent for an analyte and provided upstream from the detection zone is a mobilisable labelled binding reagent for said analyte; and wherein said second assay comprises a detection zone comprising an immobilised binding reagent for the analyte and provided upstream from the detection zone is a mobilisable labelled binding reagent for the analyte and a scavenger reagent for said analyte, wherein detection of the presence of labelled binding reagent at the detection zones provides an indication of the presence and/or extent of the analyte in the sample.

In another aspect, the invention provides a method of fabricating an assay device to reduce a hook effect, wherein the assay device comprises a first flow-path and a second flow-path, comprising the steps of providing a first flow-path comprising a porous carrier and a second flow-path comprising a porous carrier; contacting the first flow-path with a first material so as to form a first zone; and contacting the second flow-path with a first material so as to form a second zone, wherein the first zone and the second zone are separated by a distance of at least about 5 mm, and whereby the hook effect is thereby reduced. In certain embodiments, the first material contains latex.

Comparative Example 1 Preparation of an Assay Device Comprising a Single Porous Carrier Comprising a First Upstream Detection Zone for a Sandwich Assay and a Second Downstream Detection Zone for an Inhibition Assay

An assay test-strip comprising a first upstream detection zone for a sandwich assay and the second downstream detection zone for an inhibition assay and a mobilisable labelled binding reagent provided upstream from said zones was prepared as follows:

A solution of 1.5 mg/ml mAb mouse anti-human β-hCG (clone 3468, supplied in house) in PBSA buffer and 7.2 KIU/ml hCG (Scipac) in PBSA/ovalbumin was mixed for 1-hour to provide an anti-βhCG-hCG conjugate. The resulting solution was plotted in the form of a line onto nitrocellulose strips (Whatman) of dimensions 40 mm length×6 mm width, having a pore-size of 8 microns and a thickness between 90-100 microns which had been laminated to a 175 micron backing layer. The conjugate was dispensed at a concentration of 1.5 mg/ml and a rate of 1 μl/cm at the 16 mm position (distance along the nitrocellulose) using a Biodot xyz3050 dispensing platform to form a second downstream detection zone.

The first detection zone was prepared by plotting a line of anti-human β-hCG antibody (clone 3468) at a concentration of 3 mg/ml in PBSA buffer and a rate of 1 μl/cm on the nitrocellulose at the 10 mm position using the Biodot xyz3050 dispensing platform. The nitrocellulose was heated briefly to 55° C. and blocked with a solution of 1% w/w PVA in 0.05% w/w Tween-20 in pH9Tris-buffered saline/5% ethanol, followed by treatment with 2% w/w sucrose in de-ionised water. The nitrocellulose was thereafter heated briefly at 75° C.

Mouse-anti-human α-hCG mAb (clone 3299, supplied in-house) was conjugated to 400 nm blue latex polystyrene latex (Duke Scientific) in BSA/sucrose and sprayed onto a glass-fibre pad (F529-09, Whatman) at a rate of 50 g/hr and 110 mm/s. The glass fibre pad was partially overlaid onto and upstream from the nitrocellulose porous carrier.

Example 1

Assay devices were prepared in a similar manner to that of Comparative Example 1 except that the first and second detection zones were provided on respectively first and second test-strips, wherein the first and second detection zones were provided on nitrocellulose, each test-strip comprising glass-fibre sprayed with mobilisable latex labelled α-hCG antibody provided upstream from the nitrocellulose.

Preparation of the First Test-Strip

The detection zone was prepared by plotting a line of anti-human β-hCG antibody (clone 3468) at a concentration of 3 mg/ml in PBSA buffer and a rate of 1 μl/cm on the nitrocellulose at the 10 mm position using the Biodot xyz3050 dispensing platform. The nitrocellulose was heated briefly to 55° C. and blocked with a solution of 1% w/w PVA in 0.05% w/w Tween-20 in pH9Tris-buffered saline/5% ethanol, followed by treatment with 2% w/w sucrose in de-ionised water. The nitrocellulose was thereafter heated briefly at 75° C.

Mouse-anti-human α-hCG mAb (clone 3299, supplied in-house) was conjugated to 400 nm blue latex polystyrene latex (Duke Scientific) in BSA/sucrose and sprayed onto a glass-fibre pad (F529-09, Whatman) at a rate of 50 g/hr and 110 mm/s. The glass fibre pad was partially overlaid onto and upstream from the nitrocellulose porous carrier.

Preparation of Second Test-Strip

A solution of 1.5 mg/ml mAb mouse anti-human β-hCG (clone 3468, supplied in house) in PBSA buffer and 7.2 KIU/ml hCG (Scipac) in PBSA/ovalbumin was mixed for 1-hour to provide an anti-βhCG-hCG conjugate. The resulting solution was plotted in the form of a line onto nitrocellulose strips (Whatman) of dimensions 40 mm length×6 mm width, having a pore-size of 8 microns and a thickness between 90-100 microns which had been laminated to a 175 micron backing layer. The conjugate was dispensed at a concentration of 1.5 mg/ml and a rate of 1 μl/cm at the 16 mm position (distance along the nitrocellulose) using a Biodot xyz3050 dispensing platform to form the detection zone.

Mouse-anti-human α-hCG mAb (clone 3299, supplied in-house) was conjugated to 400 nm blue latex polystyrene latex (Duke Scientific) in BSA/sucrose and sprayed onto a glass-fibre pad (F529-09, Whatman) at a rate of 50 g/hr and 110 mm/s. The glass fibre pad was partially overlaid onto and upstream from the nitrocellulose porous carrier.

The test-strips according to Example 1 and Comparative Example 1 were tested using in-house readers with calibrated hCG buffer standards at concentrations 0, 25 50, 100, 250, 500, 1000, 2500, 5000, 10000, 15000, 20000, 25000, 50000, 150000, 200000 and 250000 mIU/ml hCG.

The signal intensity measured at the inhibition detection zones as a function of hCG concentration of the assays of example 1 (denoted by --♦--) and comparative example 1 (denoted by --▪--) is shown in FIG. 2 as signal in arbitrary units vs. mIU/ml hCG.

As can be seen from this Figure, the inhibition detection zone of Comparative Example 1 shows an initial plateau at levels of hCG ranging from 0-100 mIU/ml, followed by a decrease in the intensity at higher levels of hCG as expected. However, at higher levels still, the signal intensity was observed to increase. By comparison, the signal intensity of Example 1 decreases at higher hCG levels without the subsequent increase in signal intensity at higher hCG levels. As can be seen, the inhibition zone of assay device constructed according to Comparative Example 1 has a more limited range over which hCG may be measured.

Example 2 Preparation of Assay Devices Comprising a First Test-Strip Comprising a First Sandwich Assay and a Second Test-Strip Comprising a Scavenger Assay in Addition to a Sandwich Assay Preparation of the Second Test-Strip.

MAb mouse anti-human β-hCG antibody (clone 3468) at a concentration of 3 mg/ml in PBSA buffer was plotted onto nitrocellulose (of type and dimensions as that according to Comparative Example 1) at the 10 mm position at a rate of 10/cm using a Biodot XYZ3050 dispensing platform to provide a sole detection zone for the first assay.

Mouse-anti-human α-hCG mAb (clone 3299) conjugated to 400 nm blue polystyrene latex (Duke Scientific) was mixed with scavenger antibody mAb mouse anti-human β-hCG (clone 3468) at 3 mg/ml to give a final % blue latex of 3% and a final 3468 concentration of 0.075 mg/ml. The resulting mixture was sprayed at 2.02 μg/cm onto F529-09 glass fibre.

The glass fibre was partially overlaid and provided upstream from the nitrocellulose to provide the first assay test-strip.

Preparation of the First Assay Test-Strip

The first assay test strip was prepared according to the first assay test-strip according to that of Example 1.

Comparative Example 2

Assay devices constructed wherein both detection zones were provided on the same porous carrier were not able to result in the measurement of an analyte concentration over an extended analyte range.

Assay devices according to Example 2 were tested using in-house detection zone optical readers with calibrated hCG buffer standards at 12 concentrations ranging from 0-250000 mIU/ml hCG. 10 replicates per concentration level were measured giving a total number of assay devices that were tested of 120.

The signal intensity vs hCG concentration for the second assay constructed according to Example 2 is shown in FIG. 3.

The first assay test-strip according to Example 2 was able to determine the amount of hCG present up to about 400 mIU/ml before the assay curve flattened off. The second assay test-strip according to Example 2 was able to detect hCG levels of greater than about 1000 mIU/ml. Measurement of the signals at both the first and second assay test-strips enabled determination of the level of hCG between about 400 mIU/ml and 1000 mIU/ml.

The Effect of Varying the Amounts of Scavenger Antibody

Second assay test-strips according to Example 2 were prepared except the amount of scavenger antibody present was varied during preparation of the strip to give a final 3468 concentration of 0.12, 0.16, 0.2 and 0.24 mg/ml.

As can be seen from FIG. 4 increasing the amount of scavenger antibody lowers the amount of analyte captured at the detection zone.

Example 3

The aim of this Example is to assess the effect of changing secondary latex spray position on assay curve performance, with the intent of reducing the hook effect.

Materials used are as follows:

Latex 2 Reagent Composition Latex 1 (Secondary latex) Test latex   3% solids, anti β hCG 42.25 mls 42.25 mls Control latex 0.7% solids, none 16.83 mls Goat anti rabbit IgG Free antibody anti β hCG  1.16 mls  1.16 mls

Two latex sprays were generated. For Latex spray 1, the distance from base of glass fibre was held constant at 5 mm, while for Latex spray 2, the distance from base of glass fibre was set at 7, 9, 10, 11, 13, 14, or 16 mm.

As shown below, the signal was measured at 3 minutes run time as a percentage attenuation (% A) of the test line.

Distance from Base of Glass [hCG] mIU/ml Fibre (mm) 400 1000 2000 10000 50000 250000 7 17.74 29.66 39.44 49.80 48.15 30.10 9 14.49 22.54 31.07 54.90 50.98 34.23 10 15.63 23.11 32.76 59.34 61.04 42.57 11 16.68 24.62 33.11 58.65 61.47 46.54 13 13.88 20.71 29.33 57.66 62.56 47.99 14 17.35 22.82 31.87 58.40 64.42 48.82 16 14.69 19.86 28.75 56.55 63.81 49.20

Results shown above and in FIG. 5 demonstrate that if secondary latex spray position is below 9 mm, the % A signal at the higher end of the curve falls greatly (for example, signal at 250,000 mIU/ml hCG drops below signal 2000 mIU/ml). It can be concluded that increasing latex spray position to 10 mm or greater, e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or greater than 30, significantly reduces the hook effect. 

1. An assay device for determining the presence or concentration of an analyte in a liquid sample over an extended concentration range comprising a first flow-path having a sole detection zone capable of immobilising a labelled binding reagent and a second flow-path having a sole detection zone capable of immobilising a labelled binding reagent, wherein the presence of labelled binding reagent at the detection zones provides an indication of the presence or concentration of analyte in said liquid sample.
 2. The device according to claim 1 wherein the first flow-path is capable of providing an indication of the level of analyte in a first concentration range and the second flow-path is capable of providing an indication of the level of analyte in a second concentration range.
 3. The device according to claim 2 wherein the first and second concentration ranges overlap.
 4. The assay device according to claim 2 which is capable of providing an indication of the level of analyte with respect to one or more thresholds.
 5. The device according to claim 1 wherein the first and/or second flow-path comprises a porous carrier.
 6. The device according to claim 5 wherein the porous carrier is a lateral flow porous carrier.
 7. The device according to claim 1 wherein the first and/or second flow-path comprises a mobilisable labelled binding reagent for the analyte provided upstream from a detection zone.
 8. The device according to claim 1 wherein the detection zone of the first and/or second flow-path comprises an immobilised binding reagent for the analyte.
 9. The device according to claim 1 wherein the first flow-path defines a sandwich assay and the second flow-path defines a competition or inhibition assay.
 10. The device according to claim 9 wherein the first flow-path comprises a mobilisable labelled binding reagent for the analyte provided upstream from a detection zone, said detection zone comprising an immobilised binding reagent for the analyte and wherein the second flow-path comprises a mobilisable binding reagent for the analyte provided upstream from provided upstream from a detection zone, said detection zone comprising an immobilised analyte or analyte analogue for the mobilisable binding reagent.
 11. The device according to claim 9 wherein the first flow-path comprises a mobilisable labelled binding reagent for the analyte provided upstream from a detection zone, said detection zone comprising an immobilised binding reagent for the analyte and wherein the second flow-path comprises a mobilisable labelled analyte or analyte analogue provided upstream from an immobilised binding reagent for the analyte.
 12. The device according to claim 9 wherein the first flow-path defines a high analyte sensitivity assay and the second flow-path defines a low analyte sensitivity assay.
 13. The device according to claim 1 wherein the first flow-path comprises a mobilisable labelled binding reagent for an analyte provided upstream from a detection zone, wherein the detection zone comprises an immobilised non-labelled binding reagent for the analyte; and wherein the second assay comprises a mobilisable labelled binding reagent for a first binding region of the analyte and a scavenger binding reagent for the analyte provided upstream from a detection zone, wherein said detection zone is capable of binding the labelled binding reagent.
 14. The device according to claim 13 wherein the detection zone of the first or second flow-path comprises an immobilised binding reagent for the analyte.
 15. The device according to claim 13 wherein the scavenger binding reagent of the second flow-path has a higher affinity for the analyte than the mobilisable labelled binding reagent.
 16. The device according to claim 13 wherein the mobilisable labelled binding reagent and the scavenger binding reagent of the second flow-path are for respectively a first and second binding region of the analyte, the detection zone comprising an immobilised binding reagent for a second binding region of the analyte.
 17. The device according to claim 13 wherein the mobilisable labelled binding reagent and the scavenger binding reagent of the second assay are provided in the same region.
 18. The device according to claim 13 wherein the scavenger binding reagent is provided in a mobilisable form.
 19. The device according to claim 13 wherein the mobilisable binding reagents and the scavenger reagent are provided on or in a first porous carrier material and the immobilised binding reagent provided at the detection zone is provided on and/or in a second porous carrier material.
 20. The device according to claim 19 wherein the first porous carrier material is glass-fibre and the second porous carrier material is nitrocellulose.
 21. The device according to claim 1 wherein the analyte is hCG.
 22. The device according to claim 16 wherein the first binding region of hCG is the alpha sub-unit and the second binding region is the beta sub-unit.
 23. The device according to claim 1 wherein the binding reagents are antibodies.
 24. The device according to claim 1 wherein the fluid sample is urine.
 25. The device according to claim 1 wherein the binding reagent is labelled with an optically detectable particle.
 26. The device according to claim 25 wherein the optically detectable particle is a coloured polymer label.
 27. The device according to claim 1, comprising a common sample application region which serves to supply liquid sample to both flow-paths.
 28. The device according to claim 1, comprising a housing wherein the first and second assays are provided within said housing.
 29. An assay device for determining the presence or concentration of an analyte in a liquid sample comprising a first flow-path and a second flow-path wherein said first flow-path comprises a porous carrier having a detection zone comprising an immobilised binding reagent for an analyte and provided upstream from the detection zone is a mobilisable labelled binding reagent for said analyte; and wherein said second flow-path comprises a detection zone comprising an immobilised binding reagent for the analyte and a mobilisable labelled binding reagent upstream from the detection zone for the analyte and a scavenger reagent for said analyte, wherein detection of the presence of labelled binding reagent at the detection zone provides an indication of the presence or concentration of analyte in said liquid sample.
 30. The assay device according to claim 29 wherein the first flow-path defines a high sensitivity assay and the second flow-path defines a low analyte sensitivity assay.
 31. The assay device according to claim 29, wherein the analyte is hCG.
 32. The device according to claim 21, wherein: a) the first flow-path is capable of providing an indication of the level of hCG in the sample in a first range of less than or equal to a first threshold; and b) the second flow-path is capable of providing an indication of the level of hCG in the sample in a second range of above or equal to a second threshold; and c) the first and second flow-paths together are capable of providing an indication of the level of hCG in a third range of greater than the first threshold but less than the second threshold.
 33. The device according to claim 29 wherein the scavenger binding reagent has a higher affinity for the analyte than the mobilisable labelled binding reagent.
 34. The device of claim 32 wherein the first flow-path is capable of providing a positive or negative indication of pregnancy, based upon whether the level of hCG detected in the sample is respectively above or below a base threshold.
 35. An assay kit for determining the presence or concentration of an analyte in a liquid sample comprising an assay device comprising a first flow-path and a second flow-path wherein: a) said first flow-path comprises: i) a porous carrier having a detection zone comprising an immobilised binding reagent for an analyte; and ii) a mobilisable labelled binding reagent for said analyte upstream from the detection zone; and b) said second flow-path comprises: i) a detection zone comprising an immobilised binding reagent for the analyte; and ii) a mobilisable labelled binding reagent for the analyte and a scavenger reagent for said analyte upstream from the detection zone, wherein detection of the presence of labelled binding reagent at the detection zones provides an indication of the presence and/or extent of the analyte in the sample.
 36. A method of fabricating an assay device to reduce a hook effect, wherein the assay device comprises a first flow-path and a second flow-path, comprising the steps of: a) providing a first flow-path comprising a porous carrier and a second flow-path comprising a porous carrier; b) contacting the first flow-path with a first material so as to form a first zone; and c) contacting the second flow-path with a first material so as to form a second zone, wherein the first zone and the second zone are separated by a distance of at least about 5 mm, and whereby the hook effect is thereby reduced.
 37. The method of claim 36, wherein the first material comprises a particulate material.
 38. The method of claim 37, wherein the particulate material comprises latex. 